In the context of skeletal tissue repair, tissue regeneration therapy is the local application of autologous (host-derived) cells to promote reconstruction of tissue defects caused by trauma, disease or surgical procedures. The objective of the tissue regeneration therapy approach is to deliver high densities of repair-competent cells (or cells that can become competent when influenced by the local environment) to the defect site in a format that optimizes both initial wound mechanics and eventual neotissue production. For soft tissue repair, it is likely that an implant vehicle(s), will be required to 1) transport and constrain the autologous cells in the defect site and 2) provide initial mechanical stability to the surgical site. In an optimal system, it is likely that the vehicle will slowly biodegrade at a rate comparable to the production of neotissue and development of strength in the reparative tissue (1).
The tissue regeneration therapy approach contrasts significantly with more passive approaches to wound repair in which no attempt is made to deliver or to recruit reparative cells to the defect site. For example, in the case of anterior cruciate ligament (ACL) repair with synthetic (presumably "inert") polymer grafts, the healing process depends entirely on local cellular responses to initiate and control the incorporation of a permanent implant (2).
Recently, more active devices have been tested using matrix scaffolds designed to deliver and/or to direct cellular processes. These have included, for example, tendon or ACL repair (3-7), meniscus repair (8-11) and articular cartilage repair (12-15). Alternatively, the use of locally delivered peptide factors, intended to stimulate recruitment of reparative cells and their attachment and/or differentiation, have also been investigated (16-19).
In perhaps the best documented tendon repair experiments to date, Silver, Dunn and their colleagues have described extensive investigations of the performance of collagen fiber prostheses for Achilles tendon (3-5) and anterior cruciate ligament (ACL) (6,7) repair in rabbits. They report that at 52 weeks postimplantation in the Achilles tendon defect, the reconstructed tendon (prosthesis+repair tissue) was about 66% as strong as the normal tissue for all implants tested, including an autologous tendon graft and glutaraldehyde- or carbodiimide-crosslinked collagen fiber composites (5). Both the autologous implants and the carbodiimide-crosslinked prostheses were observed to biodegrade rapidly, then regain strength rapidly as new tissue was produced. Glutaraldehyde cross-linked prostheses biodegraded much more slowly in the Achilles tendon model and became surrounded by a thick capsule that eventually stopped the degradation process. While the neotendon developed in these studies was similar to normal, it was not identical. For example, the crimp angle of the neotendon collagen was similar to normal tendon in all implants, but the length of the neotendon crimp was less than about 30% of normal for the collagen prosthetic devices. In addition, the moduli of the neotendons formed from the more rapidly degrading implants (autologous tendon and carbodiimide-crosslinked collagen fibers) were significantly lower than for normal tendon. Finally, the neotendon observed did not assemble with the fascicle microarchitecture of normal tendon. These researchers conclude that the rate of degradation of the prosthesis, and the consequent transfer of load to the new tissue, may be as important as the initial prosthesis tensile strength in determining the ultimate properties of the repair tissue (5). A similar generation of neoligament was observed in the ACL implants after 20 weeks, although the recovery of strength of the tissue may be somewhat slower in the avascular synovial environment (7).
Based on this evidence, it is clear that at least in the healthy animal, repair-competent cells can be recruited from the tissues surrounding defects in tendons and ligaments, and that these cells will initiate the production of neotissue. It remains unclear from these investigations to what extent the recruited cells represented differentiated phenotypes (e.g., tendon fibroblasts), as opposed to undifferentiated pluripotent stem cells, or whether increased numbers of such cells would enhance the rate of synthesis or the microarchitecture and mechanical properties of the neotissue produced.
Many cell-mediated processes related to the production of skeletal tissue depend on the number of cells involved, both in the rate and magnitude of the effect. For example, in the in vitro production of connective tissue, the rate of collagen gel contraction by fibroblasts embedded in the gel is dependent on the number of cells present in the culture (20). A similar gel-contracting activity has also been correlated with cell density-dependent secretion of a contraction-promoting factor by endothelial cells (21). In addition, the extent of fibroblast orientation in cultures grown on collagen gels is directly related to the initial cell density (22). This cell orientation effect has been correlated with the observation of "organizing centers" in the culture, the number of which has been suggested to be a direct indicator of morphogenetic capacity at the molecular and cellular levels (23).
Cell density-dependent differentiation was clearly demonstrated in the culture of chick limb bud cells (24). When cultured at very low density (10.sup.6 cells/35 mm dish), these cells do not exhibit chondrogenic or osteogenic properties. At "intermediate" cell culture densities (2.times.10.sup.6 cells/35 mm dish), the cells exhibit the maximum frequency of osteogenesis, while at still higher density (5.times.10.sup.6 cells) the maximum frequency of chondrocyte phenotypes is observed.
In each instance cited above, the number of cells initially present strongly influences the nature of cell-mediated processes involved in skeletal tissue formation and the rate at which these developmental and physiological processes occur. Therefore, in the reparative processes of skeletal tissues, Caplan and coworkers have hypothesized that some minimum threshold of cell number may be required at the repair site before formation of "normal" neotissue can occur (25). Furthermore, in many cases, this minimum threshold may exceed the number of recruitable reparative cells, including less committed cells that can differentiate to repair competent phenotypes; therefore, the extent to which the reparative process can occur may be limited by this single parameter.
Preliminary investigations of the tissue regeneration therapy approach have recently been conducted in a tendon repair model in the Achilles tendon of the rabbit (25). There were three components to this model: the defect, the cells and the vehicle to deliver the cells to the defect site. The delivery vehicle in this model must restrain the cells at the defect site, stabilize the tissue mechanics, then slowly biodegrade as new tissue is produced.